The rapid detection of microorganisms, particularly highly virulent pathogens, is required for the timely treatment of serious infections. Contamination of air or water by pathogenic microorganisms can occur naturally, can be the result of unintended human interference, or can occur as a result of intentional use of biological warfare agents against military and civilian populations. Because of the ability of pathogens to disseminate and infect human populations rapidly, a detection system requires speed, versatility and, preferably, portability. Early detection and identification of pathogens in patients allows a health care worker to diagnose and appropriately treat a patient. Remote sampling and detection of microorganisms can limit exposure to biological agents through the identification of contaminated areas. These areas can then be quarantined and decontaminated by appropriately trained individuals.
However, in spite of the need for rapid detection of pathogens, detection equipment in current use has significant shortcomings. Manipulating and interpreting pathogen detection devices in the field is a hazardous duty, and can be made more difficult by cumbersome protective clothing worn by health care or military personnel. Thus, remote and automated sensing is required to address both safety and efficiency concerns. To be truly effective as a monitoring system, it also must be widely distributed, such that detection of bioterrorism induced or natural outbreaks can be rapidly identified and controlled. In turn, the need for a widespread early warning network demands that any detection device be accurate, automated and relatively inexpensive.
There are several methods commonly used to detect pathogens in collected samples, but not all of these methods are rapid, readily automatable or of low cost. These include (i) amplification of pathogen-specific nucleic acid sequences, including methods for amplifying pathogen-specific nucleic acid sequences requiring numerous time-consuming steps that are difficult to automate and often produce false positives or false negatives; (ii) culture of pathogens on appropriate growth media, followed by isolation and either time-consuming biochemical or histological assays; (iii) mass spectrometer-based detection of pathogen-specific components, in which each detection unit is expensive to produce; and (iv) serological-based assays, which have limited sample size and can only detect pathogens in an infected individual.
There are several disadvantages to these methods. Nucleic acid amplification and many examples of biochemical assays require a method of breaking open the cell and isolating the components. The techniques used to break open the cell have been shown to make these assays difficult to reproduce and inaccurate (Dang et al., 2001, Appl Environ Microbiol 67(8):3665-70). Culturing pathogens on solid growth media can take several days, in which time an infected patient could die or be seriously compromised. Mass spectrometry, although accurate, requires larger sample sizes, high trained operators, and a very expensive testing unit.
One general class of methods in common use for pathogen detection, enzyme-linked immunosorbent assays (ELISAs), can be made automatable, rapid and inexpensive. With regard to the latter, low cost provides multiple monitoring device capability. However, ELISAs are not very sensitive, and incapable of detecting small numbers (e.g., 1-10 anthrax spores) of pathogens. The method of the present invention combines the cost-efficiency and rapidity of an ELISA-based “dipstick” assay with the increased sensitivity of bioamplification. In this manner, the method of the invention is able to accurately and rapidly detect very small amounts of pathogens in airborne, fluid, or surface samples. The simplicity and accuracy of this protein amplification assay imparts the ability to produce relatively simple and portable detection devices.
In the case of Anthrax, treatment is effective if initiated within 72 hours of infection. This means that samples must be analyzed in time to identify potentially infected individuals and begin treatment. The invention discloses a device that takes advantage of this latency period to improve on the accuracy of an inexpensive test, significantly reducing false positive and false negative results using bioamplification. This method and device for detection of pathogens such as Anthrax can give automated and accurate pathogen detection within 12-24 hours, leaving plenty of time to begin treatment for infected individuals.